Production of the key Protective Antigen (PA) protein of the current licensed anthrax vaccine is achieved in avirulent strains of the native organism, Bacillus anthracis. While alternative expression systems (e.g., E. coli) have been used in anthrax vaccine production, there are several reasons that B. anthracis continues to be used. In the current reporting period, we collaborated with the Biotechnology Core Unit of NIDDK to explore ways to further enhance yields of proteins from the protease-deficient strain BH500, which we previously developed. RNA-seq was performed on strain BH500 producing recombinant PA (rPA) or containing an empty expression vector, with the objective of identifying stress responses that could point to genes and pathways that might be genetically modified to enhance protein production. The bacteria were grown in a bioreactor like that used in large scale protein production. Samples were collected at three stages during the growth phase and RNA-seq data generated. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase sigma factor 54, and sigB, encoding the general stress transcription sigma factor B. Among many other changes in expression observed, it was particularly interesting to find large changes in the extracellular chaperones csaA and prsA. The prsA genes encode foldases, cell-surface lipoproteins that have proline-peptide isomerase activity. This activity is essential to allow the proline isomerization needed to properly fold proteins as they are secreted through the Gram-positive cell wall. Prior evidence had suggested that a principal limitation on production of rPA did not lie at the level of protein expression but in the capacity of the secretion machinery, of which the foldases are key components. It follows that increasing the amount of the foldases may enhance rPA yields. In fact, this has been shown for other Gram-positive bacteria. Therefore we plan to modify our BH500 strain to express more of the foldases. The resulting host strains may also be useful in production of heterologous (i.e., non-bacterial) proteins for use in various other ongoing projects. In other work during fiscal year 2019, we extended our analysis of the central transcriptional activator of B. anthracis, AtxA. Methods were developed for production and purification of the protein from E. coli. Mutated proteins were produced that are altered in two histidine residues that other groups have shown to be phosphorylated. Biophysical characterization of the proteins has been done as a basis for future studies to identify how the protein recognizes the genes that it controls.